Project Summary. There remains a critical need to stop the spread of HIV-1, which still infects 6000 persons per day worldwide, and better therapies to treat or even cure established infections would be beneficial. Broadly neutralizing antibodies (bNAbs) are now being evaluated in passive immunization for preventing and treating HIV-1 infection; and eliciting bNAbs is a central goal of multiple vaccine- development efforts. All of these various translational programs will benefit from an expanded mechanistic and quantitative knowledge of how neutralizing bNAbs act. There are many gaps in our knowledge about how bNAbs interact with HIV-1 in vivo, in environments where neutralization in the absence of activating Fc-receptor interactions is insufficient to potently prevent transmission or suppress viremia. NAbs are traditionally evaluated for potency, but other, relatively neglected, aspects of neutralization may also influence the outcome of Env vaccination and bNAb-based therapy. Our long-term goal is to be able to identify bNAbs or combinations of bNAbs with optimal anti-viral properties and thereby to minimize the risk of viral breakthrough and escape. Our overall objective is to quantify and explain neglected aspects of HIV-1 neutralization that may improve the designs of bNAb-based vaccination and therapy. Our central hypothesis is that the efficacy of neutralization (the converse of persistent infectivity), the varying interactions between cells and virions, the mechanisms and kinetics of how antibodies impede viral entry, and the propensity for escape together influence the outcome of passive and active immunization. Our observations of markedly reduced maximum neutralization plateaus for some HIV-1 strains when they are treated with certain NAbs or with sera from trimer-immunized animals are pertinent to this hypothesis. In summary, by quantifying the effects of NAbs on HIV-1 entry into different cells, and by exploring the conditions of viral escape, we seek to complement simple measurements of neutralization potency and thereby build an explanatory predictive model for how NAbs act in vivo. An emphasis will be to use, whenever justified, clinically relevant bNAbs as key tools in our in vitro research, to maximize the translational potential of the new knowledge we will generate. To test our central hypothesis and thereby obtain our objectives we propose three Specific Aims: 1. To define the causes of the persistent fraction in HIV-1 neutralization. 2. To determine the dynamics of HIV-1 neutralization. 3. To explain neutralization escape mechanisms in vitro and in vivo. We expect our studies to bridge gaps in knowledge of how bNAbs act in vitro and in vivo and hence guide improvements to clinically relevant passive immunization and active vaccination programs.